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1.4: What's in a developmental biologist's "toolbox"?

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    27191
  • Although developmental biology encompasses the study of many different types of organisms, we will see that many studies use the same types of experiments to answer similar questions. 

     

    In Situ Hybridization: where is an RNA expressed?

    We know that within an embryo, all of the cells share the same genome. However, we know that all the cells are not expressing all the genes. In situ hybridization (ISH) is a method used to visualize where an particular RNA is expressed. In developmental biology ISH is often performed on whole embryos (sometimes abbreviated WISH), but it is also possible to perform on sections of embryos or tissues. 

    In the above video, a chemical called digoxigenin (abbreviated DIG) labeled the RNA probe. This chemical can be detected with an Anti-DIG antibody that is fused with an enzyme called alkaline phosphatase (AP). This enzyme catalyzes a reaction that produces a blue/purple product from the chemical BCIP/NBT. 

    Global gene expression analysis: what are all the RNAs being expressed?

    In situ hybridization is an essential tool, but is typically limited in focus to examining a small number of genes. Since the late 1990s, developmental biologists have isolated mRNA from embryos to compare the effects of time, stage, mutations, or treatments on gene expression at a global scale. Microarrays, in which samples of RNA were fluorescently-labeled and hybridized slides containing DNA spots for each gene in the genome, where one of the first tools that made this possible.  The increase in speed, ease, sensitivity, and affordability of sequencing RNA (technically cDNAs produced from mRNA are sequenced) has made examining gene expression at a genomic scale possible.

    More recently, the The journal Science named  single cell transcriptomics the Breakthrough of the Year for 2018 (https://vis.sciencemag.org/breakthrough2018/finalists/#cell-development).

    Immunohistochemistry / Immunofluoresence: where is a protein present?

    The previous experiments looked only at mRNA expression. If the gene is also regulated post-transcriptionally, the protein may not exhibit the same expression pattern as the RNA. Antibodies are proteins that recognize and bind to other proteins, just as they do in the immune system. In these experiments, an antibody that recognizes the protein of interest applied to the embryo. Often to amplify the signal, a "secondary antibody" is applied next. The secondary antibody might either be coupled to an enzyme that catalyzes a reaction (similar to ISH) or labeled with a fluorescent dye. Fluorescent tags of two (or more) different wavelengths can be used to show colocalization (overlap) between two proteins. 

    Transplantation: if I move or remove cells, will cell fates change?

    Developmental biology has its roots in embryology. Long before the molecular era, scientists manipulated embryos by removing or separating cells, changing their position within embryos, and even transplanting cells between embryos of different species. Transplantation experiments are often used to uncover whether cell fates are already determined or can be influenced by signals from the environment.  

    RNA interference: does inhibiting a gene product affect development?

    Andrew Fire and Craig Mello won a Nobel Prize for their discovery and characterization of RNA interference (RNAi) in C. elegans. In RNAi, small RNAs that are complementary to an RNA "interfere" with translation, effectively reducing or eliminating the amount of functional protein present. A small RNA that functioned in a naturally-occurring gene regulation mechanism was first discovered in C. elegans (Lee et al., 1993), but Fire and Mello performed crucial experiments that showed that exogenously-supplied double-stranded RNA could be used to manipulate gene expression in the worm. Now developmental biologists have the ability to easily reduce protein levels without creating genetic mutants, which is a much more time-consuming process (especially prior to gene-targeting mechanisms such as CRISPR). 

    Genetic mutants

    Although RNAi has made exploring the functions of genes faster and easier, ultimately it is genetic mutants (changes in DNA sequence) that best demonstrate the function of an individual gene. Whether or not these mutants occur naturally, are identified in forward genetic screens, or are produced using modern gene targeting methods, mutants tell us what effect a gene has on development, by showing us what changes occur when the gene is lost or altered.

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